2-formylmercaptobenzothiazole

ABSTRACT

There is disclosed a novel formylating agent, 2-formylmercaptobenzothiazole.

This is a Divisional of U.S. application Ser. No. 08/138,558 filed Oct.15, 1993, now U.S. Pat. No. 5,442,047, which in turn is a continuationof U.S. application Ser. No. 07/777,385 filed Dec. 4, 1991, nowabandoned, which was the United States national applicationcorresponding to International Application No. PCT/US90/03328 filed Jun.19, 1990 and designating the United States, which PCT application is inturn a continuation of U.S. application Ser. No. 369,578 filed Jun. 21,1989, now abandoned, the benefit of which applications is claimedpursuant to 35 U.S.C. 120, 363, and 365(c).

This invention relates to a novel process for converting gentamicin B toisepamicin, 1-N-[(S)-3-amino-2-hydroxypropionyl] gentamicin B and to anovel formylating agent, 2-formylmercaptobenzothiazole, useful in theprocess.

More particularly, this invention relates to a process for convertinggentamicin B to 3,6'-di-N-formylgentamicin B by using2-formylmercaptobenzothiazole, acylation of the 1-amino group with an(S)-isoserine derivative, followed by removal of the protecting groupsunder conditions which result in high yields of the desired product.

Isepamicin, which has the formula ##STR1## is a known aminoglycosideantibiotic. The preparation of this compound from gentamicin B isdescribed in U.S. Pat. No. 4,230,847. The process described in thepatent involves forming 3,6'-di-N-benzyloxycarbonyl gentamicin B byreacting a cuptic-nickel (II) salt complex of gentamicin B withN-benzyloxycarbonyloxy-phthalimide and then reacting the intermediatecompound with N-(S-3-benzyloxycarbonylamino-2-hydroxypropionyloxy)succinimide. The benzyloxycarbonyl protecting groups were removed fromthe resulting material by catalytic hydrogenation overpalladium-on-carbon to produce isepamicin in 60% yield from theintermediate starting material.

Tsuchiya, et al., Tetrahedron Letters No. 51, pp 4951-4954 (1979),describe a complex multistep process for the protection of the3,3",6'-amino groups in Kanamycin A comprising zinc acetate chelation,3,6'-N-bisbenzyloxycarbonylation, zinc removal, carbonate formation, andfinally trifluoroacetylation of the 3"-amino group. The3,3",6'-N-triblocked Kanamycin A so produced is then acylated at thefree C-1 amino group using an active ester of1-N-[(S)-4-benzyloxycarbonylamino]-2-hydroxybutyric acid. Finally theresulting product is subjected to a 2-part deprotection scheme to giveAmikacin. A similar sequence is also described for the conversion ofdibekacin to its 1-N-[(S)-4 -amino-2-hydroxybutyryl] derivative. Noreference to the use of this process for the selective acylation ofother aminoglycosides was disclosed. The Tsuchiya et al. processsequence is cumbersome involving both trifluoroacetylation andbenzyloxycarbonylation in the protection steps and requiring bothaminolysis and hydrogenolysis in the deprotection steps. These stepsrender the process commercially unattractive both in the sense ofoperating costs and capital requirements for implementation.Furthermore, in our hands, and contrary to the implications in theTsuchiya et al. process description, zinc acetate chelation does notinvariably lead to selective 3,6'-diblockade in aminoglycosides otherthan Kanamycin and dibekacin. Thus, unexpectedly, the zinc acetatechelation of gentamicin B followed by acylation with formylimidazoleleads primarily to 1,6'-N-diformylation and not 3,6'-diformylation.Again acylation of this same gentamicin B zinc acetate chelate with adifferent formylating agent, formylacetic mixed anhydride, gives rise,in addition to the desired 3,6'-N-diformyl-gentamicin B, to undesiredlevels of acetylated gentamicin B products. To underline the difficultyof prediction, formyl p-nitrobenzoic mixed anhydride provedinsufficiently reactive to be useful in the formylation of gentamicin. Bzinc acetate chelate, whereas use of formyl p-anisic mixed anhydrideafforded excellent yields of the desired 3,6'-diformyl gentamicin B,contaminated with minor amounts of anisoyl impurities.

Use of metal acetates, for example, zinc acetate and the like, give riseto small amounts of undesirable by-products, for example N-acetylderivatives, which are difficult to remove and decrease the yield ofdesired product.

The formylation of aminoglycosides has been previously described byThomas et al., Tetrahedron Letters, Vol. 21, pp 4981-4984 (1980) inconnection with the preparation of 1-N-alkylated Kanamycin antibiotics.However, Thomas et al. do not teach the value of formylation as anamine-protecting group in producing 1-N-acylated aminoglycosides. Nor isthis point recognized in any other aminoglycoside literature. Instead,the literature describes the use of such as trifluoroacetyl,trichloroacetyl, and phthaloyl groups for the protection of amine groupsand teaches that the aminolysis or hydrazinolysis of such groups can becarried out without materially affecting certain other N-acyl groupswhich may be present on the molecule. In short the selective aqueousbase hydrolysis of formyl groups from 3,6'-N-formylated-1-N-acylatedaminoglycosides is unprecedented.

We have now found that a novel formylating agent2-formylmercaptobenzothiazole of formula II, ##STR2## can selectivelyformylate the 3, 6'-amino groups of the gentamicin B zinc chelate.Moreover, the procedure of this invention result in a high yield if oneuses a different zinc salt, such as zinc pivaloate, to avoid formationof undesirable by-products. Furthermore, we have found that a3,6'-N-diformyl gentamicin B obtained, by removing zinc from thechelate, can be selectively acylated with N-formyl-(S)-isoserine activeester only at the C-1 amino group without separate protection of theC-3" methylamino group as in the Tsuchiya et al. procedure. Finally, wehave found that all formyl groups can be removed from the resulting3,6'-N-diformyl-1-N-[N-formyl-(S)-isoserinoyl]-gentamicin B by aqueousbase hydrolysis in high yield without removing the desired isoserineside chain.

SUMMARY OF THE INVENTION

This invention relates to an improved multistep process for convertinggentamicin B to isepamicin in high yields.

The process of this invention comprises

(a) reacting gentamicin B with a chelating agent and then with2-formylmercaptobenzothiazole capable of selectively introducing formylgroups in

gentamicin B to form 3,6'-di-N-formylgentamicin B.

(b) acylating the 1-amino group of 3,6'-di-N-formylgentamicin B with anactivated N-acyl protected (S)-isoserine compound;

(c) removing all protecting groups; and

(d) isolating isepamicin.

DETAILED DESCRIPTION OF THE INVENTION

The intermediate compound, 3,6'-di-N-formylgentamicin B, is prepared byreacting a divalent metal salt complex of gentamicin B with2-formylmercaptobenzothiazole to introduce formyl protecting groups atthe 3,6'-positions. The metal salt complex is prepared using methodsdisclosed in U.S. Pat. No. 4,136,254 and Thomas, et al., TetrahedronLetter, Vol. 21, 4981-4984 (1980).

The reaction scheme for preparing 3,6'-di-N-formylgentamicin B (III) isset forth below: ##STR3##

Transition metal salts useful as complexing agents in the process ofthis invention include such divalent salts as copper (II), nickel (II),cobalt (II), cadmium (II) and zinc (II) as well as mixtures thereof. Thedivalent metal salts are salts of organic acids, preferably organicacids such as formic, acetic, propionic, pivalic and benzoic acid.Preferred divalent metal salts include pivaloate salts of zinc (II) andcobalt (II). Of particular use is zinc (II) pivaloate.

The formation of the divalent salt complex of gentamicin B is carriedout in an inert organic solvent. Preferred organic solvents are forexample, dimethylsulfoxide, dimethylformamide, dimethylacetamide,methylene chloride, toluene, ethyl acetate and mixtures thereof.

In preparing the divalent salt complex of gentamicin B, it has beenfound advantageous to employ from about 1.5-4.5 moles of the divalentsalt, for example zinc (II), per mole of gentamicin B. The preferredmolar ratio of reagents is about 2.7-3.5 moles of divalent salt per moleof gentamicin B.

The divalent salt complex of gentamicin B is reacted with2-formylmercaptobenzothiazole which introduces a formyl protecting groupat both the 3 and 6'-amino groups.

The molar quantity of 2-formylmercaptobenzothiazole is usually 2-3 to 1of the molar quantity of the divalent salt complex of gentamicin B. Thepreferred molar quantity is 2.5 to 1.

Formylation of this divalent salt complex of gentamicin B is carried outat a temperature of from 0° C. to 40° C., preferably from 20° C. to 30°C.

The formylation reaction of the divalent salt complex of gentamicin B isconveniently carried out in an organic solvent or a mixture of organicsolvents. Organic solvents that can be utilized in this reaction includedipolar aprotic organic solvents, for example, dimethylsulfoxide,dimethyl formamide, dimethyl acetamide, and the like. It has also beenfound advantageous to employ mixtures of a dipolar aprotic organicsolvent with an inert organic solvent, for example, toluene, ethylacetate, 1,2-dimethoxyethane, tetrahydrofuran, acetonitrile, methylenechloride, and the like. A preferred mixture of solvents is dimethylsulfoxide with either methylene chloride or ethyl acetate.

While all prior processes require the use of a precipitating agent or aprocedure to remove the divalent metal salt cation, the use of2-formylmercaptobenzothiazole and zinc allows for an extractive removalof the zinc 2-mercaptobenzothiazole salt in the organic solvent layer.

The aqueous solution comprises 3, 6'-di-N-formylgentamicin B in a yieldof approximately 90-95%. The product can be isolated and purified byconventional methods such as ion exchange chromatography.

Introduction of the (S)-isoserine side-chain at the 1-amino group of3,6'-di-N-formylgentamicin B is carried out by means of in-situ activeester formation of the N-protected-(S)-isoserine with an activatingreagent in the presence of dicyclohexylcarbodiimide according to thefollowing reaction scheme: ##STR4##

N-Protected -(S)-isoserine compounds that are useful in the process ofthis invention are hose wherein the amino group of -(S)-isoserine isprotected with an acyl group which can be easily removed underconditions which remove formyl protecting groups and which will notaffect other portions of the molecule. Acyl protecting groups which canbe easily removed under mild basic conditions or with hydrazine areutilized in the process. Examples of N-acyl protecting groups which areeasily removed under mild basic conditions include formyl,trichloroacetyl and trifluoroacetyl. Examples of N-acyl protectinggroups which are easily removed by hydrazine include phthaloyl andsuccinoyl. The preferred N-acyl protecting group for the isoserinecompound is the formyl group.

N-Protected isoserine compounds that are useful in the process of thisinvention include N-formyl-(S)-isoserine, N-phthaloyl-(S)-isoserine;N-trichloroacetyl-(S)-isoserine, and N-trifluoroacetyl-(S)-isoserine.The preferred N-protected isoserine compound is N-formyl-(S)-isoserine.

Active esters of N-protected-(S)-isoserine are prepared by reacting theisoserine compound with a compound such as N-hydroxybenzotriazole,N-hydroxy succinimide, imidazole, N-hydroxyphthalimide,N-hydroxy-5-norbornene-2,3-dicarboximide and the like, in the presenceof a coupling agent such as dicyclohexyl-carbodiimide.

The reaction of N-protected -(S)-isoserine with 3,6'-di-N-formylgentamicin B is carried out at temperatures between 0° C. and 40° C.,preferably at about room temperature, in a solvent. Examples of solventswhich can be employed in the process of this invention include proticorganic solvents, for example alcohols, such as methanol, ethanol,propanol and the like; mixtures of water and alcohol, such as aqueousmethanol, aqueous ethanol, and the like; aprotic solvents, such asdimethyl formamide, dioxane, methylene chloride. A preferred solvent isaqueous methanol.

The compound obtained by reacting N-formylisoserine with3,6'-di-N-formyl gentamicin B is triformylisepamicin, compound IV.

The protecting groups are removed from the Compound IV by hydrolysisaccording to the following reaction scheme. ##STR5##

Prior to deblocking Compound IV, the solvent is removed from thereaction mixture. Although deblocking by hydrolysis is a conventionalprocedure the specificity for formyl group removal without removal ofthe isoserine side chain is unprecedented in the aminoglycoside field.It has been found that when the hydrolysis reaction is conducted bystirring overnight at room temperature, an excellent yield (88-90%) ofdesired product is obtained. The resulting hydrolysate is acidified topH 6 with acid and isepamicin is obtained by isolation.

The following Examples are illustrative of a preferred mode of carryingout our invention but are not to be construed as limiting the scopethereof. Equivalents thereof will be obvious to one skilled in the artreading this application and said equivalents are contemplated asincluded within this invention. In the example HPLC means HighPerformance Liquid Chromatography; Amberlite IRC-50 is a weak cation ionexchange resin available from Rohm and Haas Company.

EXAMPLE 1 Preparation of 2-Formylmercaptobenzothiazo

To a dry three-necked 500 ml round bottom flask was added 80 ml ofacetonitrile, 5.0 ml (0.133 mole) of formic acid and 18.1 g (0.266 mole)of sodium formate. The resulting suspension was cooled to 0°-5° C. and14.6 ml (0.2 mole) of acetyl chloride was added slowly, whilemaintaining the temperature of the reaction mixture below 8° C. Afterthe addition of acetyl chloride was complete, the reaction mixture wasallowed to warm up to 18°-20° C. The completeness of the reaction wasjudged by ¹ H-NMR. To the heterogeneous mixture containing acetic formicanhydride, 60 ml of acetonitrile was added followed by 20 g (0.103 mole)of 2-mercaptobenzothiazole and the temperature was allowed to warm up to32° C. and maintained at that temperature, while the progress of thereaction was monitored by HPLC at 10 minute intervals. The reaction wasconsidered to be complete when ca. 4% of 2-mercaptobenzothiazole (byarea %) remained unreacted or when its area % starts to increase due todecomposition of the product.

The reaction mixture was then quenched with 200 ml of ice-water andstirred for 2 minutes. The precipitated product was filtered, washedthoroughly with water (4×150 ml) and dried under vacuum until the watercontent of the solid is <0.08% to afford 21.4 g (98% pure by HPLC, 89%yield) of 2-formylmercaptobenzothiazole, m.p. 125°-130° C.(decomposition)

¹ H-NMR (CDCl₃) w 7.36-7.44 (m,3H), 8.45-8.52 (m, 1H), 9.92 (S,1H).

EXAMPLE 2 Preparation of Zinc Pivaloate

To 250 ml of water, warmed to 60°-70° C., was added 56.1 gm (0.55 mole)of pivalic acid (trimethylacetic acid). Then 31.25 gm (0.25 mole) ofzinc carbonate was added portionwise over a period of 10 to 15 minutesand then the temperature was raised to 96°-98° C. After agitating thereaction mixture for 1 hour, the mixture was cooled to 4° C. with an icebath for 30 minutes and the suspension filtered. The filter cake waswashed once with 75 ml cold water and 3×50 ml cold acetone. Theresulting product was dried at 60° C. for 16 hours in a draft oven toyield 58 gm (87%) of zinc pivaloate.

EXAMPLE 3 3,6'-Di-N-formylgentamicin B

To 285 ml of dimethylsulfoxide and 285 ml of methylene chloride wasadded 34.0 g (127 mmole) of zinc pivaloate and 19 g of gentamicin B(purity 93.1%, 36.7 mmole). The resulting suspension was stirred for 10to 15 minutes at room temperature to effect solution. To this solutionwas added 16.0 g of 2-formylmercaptobenzothiazole (81.9 mmole) and afterfive minutes an aliquot was taken for liquid chromatographic analysis ofthe ratio of the monoformyl/diformyl peaks. Two more small additionswere made such that the eventual total charge was 16.95 g (86.8 mmole)to give a final peak ratio of 0.02, which was judged to be complete.

The reaction mixture was transferred to a 2 liter separatory funnel and800 ml of water was added. The phases were separated, and the aqueouslayer was re-extracted with a 30 ml portion of methylene chloride. Theaqueous layer was then filtered thorugh a small pad of celite to removea haze of solids.

The filtrate was diluted with water to a final volume of 2 liters, andits pH was about 6 at this point. This aqueous solution was charged ontoa column containing 800 ml of Amberlite IRC-50 resin which had beenadjusted to a partial ammonium cycle. The product was eluted with 0.75Nammonium hydroxide; the fractions containing the product were pooled andconcentrated to yield a solution, which was assayed by liquidchromatographic analysis and found to contain 17.9 g (90.5%) of3,6'-di-N-formylgentamicin B. Mass spectrum m/e (%) (FAB/GLY-THIO) 539(100, M⁺ +1), 511(9), 380(9), 350(4), 191(10), 190(5), 160(28).

¹ H-NKR (400 MHz, D₂ O; pH=9) w 1.25 (s, 3H, C-4"--CH₃), 2.57 (s, 3H,N--CH₃), 5.11 (d, J=4.02 Hz, 1H, anomeric), 5.38 (d, J=4.02 Hz, 1H,anomeric), 8.15 (s, 1H, N--CHO), 8.16 (s, 1H, N--CHO).

¹³ C-NMR (100 MHZ, D₂ O; pH=9) w 51.36(C-1), 47.8(C-3), 38.96(C-6'),64.5(C-3"), 37.01(N--CH₃), 22.22(C-4"--CH₃), 165.47 (N--CHO),164.76(N--CHO).

EXAMPLE 4 Preparation of N,O-Diformyl-(S)-Isoserine

To a one liter round bottom flask containing 50 g of (S)-isoserine(0.476 mole) and 62.5 ml of formic acid was added in 30 min. a freshlyprepared acetic formic anhydride solution (5 eq.) at 0°-5° C. Afterexamining the completion of reaction by H¹ -NMR (approx. 2 hrs), themixture was concentrated under vacuum at 40° C. to half of the originalvolume. 250 ml of isopropanol was added slowly with simultaneous coolingto effect crystallization. The slurry was stirred at 0° C. for one hour.The product, N,O-diformyl-(S)-isoserine, was filtered and washed withisopropanol. This afforded 64 g of N,O-diformyl-(S)-isoserine; 84%yield; m.p. 139.5°-141.5°; [a]_(D) ²⁰ : -38° (1%, MeOH). 6 * Aceticformic anhydride was prepared by adding acetyl chloride to 1.2 eq. ofsodium formate (anhydrous, micronized) in anhydrous acetonitrile (theconcentration of sodium formate/CH₃ CN can be as high as 50%) at 0°-5°C. The reaction takes 2 hours to complete. The precipitate was filtered,the filtrate was used as is in the above reaction. Some carbon monoxideis evolved from this mixture, depending on the temperature. Reasonablestability was observed at 0° for one month.

¹ H-NMR (D₂ O) w 3.8 (dd, 1H, J=14.6, 4.4 Hz), 3.91 (dd, 1H, J=14.6, 5.5Hz), 5.38 (dd, 1H, J=5.5, 4.4 Hz), 8.15 (s, 1H), 8.27 (s, 1H).

EXAMPLE 5 Preparation of N-Phthaloyl-(S)-Isoserine

To a stirred suspension of 15.75 g (150 mmole) of (S)-isoserine and 22.2g (150 mmole) of phthalic anhydride in 600 ml oftoluene:dimethylformamide (3:1), was added 2.1 ml (15 mole) oftriethylamine. The suspension was heated to reflux and the watergenerated was removed using a Dean-Stark condenser. No additional waterseparated after two (2) hours at reflux. The solvent was evaporated to afinal volume of approximately 100 ml. The reaction mixture was cooled,diluted with ice-water and acidified with 2N hydrochloric acid to afforda precipitate. The product was filtered, washed with ice-water and driedunder vacuum to yield 30.4 g (86%) of N-phthaloyl -(S)-isoserine; m.p.227°-228° C; [a]_(D) ²⁰ : +10 (1%, DMF). ¹ H-NMR (DMSO-d₆) w 3.76 (dd,1H, J=13.46, 7.69 Hz), 3.84 (dd, 1H, J=13.46, 5.77 Hz), 4.3 (dd, 1H,J=7.69, 5.77 Hz), 7.77-7.89 (m, 4H).

EXAMPLE 6 Preparation of N-trifluoroacetyl-S-isoserine

To a stirred solution of sodium methoxide in methanol, 11 ml (1 eq.,24.8% w/w solution) was added 5 g of (S)-isoserine. The mixture wasstirred at room temperature for 15 minutes until a homogeneous solutionwas obtained. Ethyltrifluoroacetate, 7 ml (1.25 q.) was added. Themixture was stirred for 30 minutes after the addition. The completenessof the reaction was monitored by ¹ H-NMR. The mixture was concentratedunder reduced pressure to as low a volume as possible. To the residue 50ml ethylacetate was added. The mixture was cooled to 0°-5° C., 25 ml of2N HCl (1 eq.) was added, followed by 5 g of solid sodium chloride. Theorganic layer was separated. The aqueous layer was reextracted with 50ml of ethylacetate. The combined organic extracts were dried (over 5 gof anhydrous magnesium sulfate), filtered and concentrated under reducedpressure to 20 ml. To it 50 ml heptane was added with stirring in anice-bath for 30 minutes. The product was filtered and dried to yield8.86 g (93%) of N-trifluoroacetyl-(S)-isoserine; m.p. 142°-143° C.;[a]_(D) ²⁰ : +12.4 (1%, H₂ O); ¹ H-NMR (D₂ O) w 3.78 (d, 2H, J=5.48 Hz),4.53 (t, 1H, J=5.48 Hz).

EXAMPLE 7 Preparation of Isepamicin

The following methods illustrate the preparation of Isepamicin.

Method A:

A stock solution of N-formyl-(S)-isoserine was prepared by stirring 20 g(124.2 mmoles) of N,O-diformyl-(S)-isoserine in a mixture of methanol(85 ml) and pyridine (15 ml, 1.5 equiv.) at room temperature for 14-16hrs. The completion of the reaction was judged by ¹ HNMR.

In a separate flask, 20 g of aq. concentrate (4.424 g active, 8.2 mole)of 3,6'-diformyl gentamicin B and 1.26 g (8.26 mmole) of1-N-hydroxybenzotriazole monohydrate were dissolved in 40 ml ofmethanol. To the stirred mixture the above N-formyl-(S)-isoserinesolution in methanol (22.2 ml 24.4 mmole, 3 equiv.) and a solution ofdicyclohexyl carbodiimide (5 g, 24.3 mole, 3 equiv.) in 20 ml ofmethanol were added simultaneously over a period of 40 min. The mixturewas stirred for 15 mins. after the addition was complete. The progressof the reaction was monitored by either HPLC or by TLC. The solventswere then removed under reduced pressure, and the product,triformylisepamicin, was hydrolysed by stirring at room temperature for16 hrs. with 90 ml of 2N NaOH. The reaction mixture was neutralised topH=6 with acid, filtered and the filtrate was diluted to a precisevolume of 1000 ml. External standard HPLC assay of this solutionindicated an 89% yield of isepamicin (4.17 g, 7.3 mole).

Method B:

A solution was prepared by dissolving 1.156 g (96.6% pure, 2.07 mole) of3,6'-di-N-formylgentamicin B, 800 mg (1.7 eq.) of N-phthaloylisoserineand 365 mg (1.2 eq.) of N-hydroxybenztriazole monohydrate in 40 ml ofmethanol. To this solution was added 700 mg (1.7 eq.) ofdicyclohexylcarbodiimide. The reaction was stirred at room temperaturefor one hour and 160 mg of N-phthaloyl-(S)-isoserine and 140 mg ofdicyclohexylcarbodiimide was added and the reaction was allowed to stirat room temperature for approximately three (3) hours. The progress ofthe reaction was monitored by TLC. The solvent was removed byevaporation and the residue taken up in 50 ml ethanol and 5 ml water.The protecting groups were removed by treating the resulting mixturewith 6.0 ml (85%) hydrazine hydrate. The reaction was heated at 85°-90°C. under nitrogen for 14 hours. External standard HPLC assay of thereaction indicated a yield of 89% (1.05 g, 1.85 mole) isepamicin.

Method C:

To 48.9 g of aq. concentrate (8.45 g active, 15.7 mmole) of3,6'-diformylgentamicin B, 2.4 g (15.7 mmole) of1-N-hydroxybenzotriazole monohydrate was added followed by 80 ml ofmethanol. To the stirred mixture, 9.5 g (47.3 mmole, 3 equiv.) ofN-trifluoroacetyl-(S)-isoserine in 40 ml of methanol and 9.7 g (47.1mmole, 3 equiv.) of dicyclohexyl carbodiimide in 40 ml of methanol wereadded simultaneously over a period of 40 min. The mixture was stirredfor 15 min. after the addition was complete. The progress of thereaction was monitored by either HPLC or TLC. The solvents were thenremoved under reduced pressure, and the product was hydrolysed bystirring with 170 ml of 2N NaOH at room temperature for 16 hrs. Thereaction mixture was neutralised to pH=6 with acid, filtered and thefiltrate was diluted to a precise volume of 1000 ml. External standardHPLC assay of the solution indicated an 88% yield of isepamicin (7.84 g,13.8 mmole).

We claim:
 1. The compound 2-formylmercaptobenzothiazole.